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South African Society of Dairy Technology Symposium 24-26/04

UNISENSOR

Unisensor is a Belgian company researching, developing and manufacturing diagnostic tools for the detection of contaminants present in food products.

EU RESEARCH PROGRAMS

2015

“ GOOD FOOD ” “ CONFFIDENCE ”

Company Timeline milestones

2005

Twinsensor PATENTED CONCEPT

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BEADYPLEX

R&D EFFORT

EXTENSO

2017

REVOLUTIONARY MILK ANALYSIS PLATFORM

MULTIPLE: + 100 molecules in a single test Detection level at EU MRL

X 17

Our Product Development Strategy In two words…

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Multiple tests in one (antibiotics)

• Reliable & Usable everywhere
• Rapid & Cost-effective
• Validated by external parties

MULTIPLEXING + CONTAMINANTS 1 x 17 x

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BEADYPLEX Scheme of analytical methods for antibiotic residues analysis

Confirmation Screening

IMMUNOAssays Structure ELISA LFIA LC-MS PHYSICO CHEMICAL Composition

TESTING PRINCIPLES METHODS

AVAILABLE MICROBIOLOGICAL Activity Inhibition

Complexity Price

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BEADYPLEX Unmet needs for a screening test

Adding Multi-Matrix dimension

• 1. Multiple families ID in a single test (vs

ELISA)

• 2. Sensitive for all/most residues (vs

microbiological methods)

• 3. High Throughput (+100 samples/day)
• 4. Cost-effective and time-saving

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BEADYPLEX - “All-In-One” screening Flow Cytometric Immunoassays – Assay principle Immunoanalysis: detection of interaction events

(receptor & antibodies).

Flow Cytometry: multi-parametric characterization

• f microparticles (beads) by lazer.

1. Different sizes 2. Different Internal fluorescence intensities

Multiplexing via microparticles characterized by:

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FCIA: simultaneous detection of residues of antibiotics from 10 families in one sample in one analysis

10 different ELISA kits combined in one test

BEADYPLEX - “All-In-One” screening Flow Cytometric Immunoassays – Assay principle

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BEADYPLEX - “All-In-One” screening Flow Cytometric Immunoassays – Assay principle

FLOW CYTOMETRIC COMPETITIVE IMMUNOASSAY

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BEADYPLEX - “All-In-One” screening Flow Cytometric Immunoassays – Assay principle

BL RL

Classification of particles Determination of assay signal intensity

Size Internal fluorescence

Flow cytometer (NovoCyte 2000, ACEA Biosciences)

Automatic Reading

3 4

Automatic Reporting

BEADYPLEX Workflow for Antibiotic residues

1

Sample preparation

2

Immunoassay

BEADYPLEX Analytical performance – Validation

Following CRLs Guidelines for the validation of Screening methods for residues of veterinary medicines (20/01/2010) based on Commission Decision 2002/657/EC Specificity/selectivity Cut-Off values Detection capabilities False positive rates Robustness Applicability

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BEADYPLEX Analytical performance

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Detection Limits (CCβ)

✓CCβ determined for all detected molecules ✓Most of CCβ ≤ MRL CCβ (β error ≤ 5%) → concentration for each antibiotic recognized by the FCIA for which at least 95% of the samples fortified at this level are identified as “screened positive” according to the established assay cut-off values

BEADYPLEX Analytical performance

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Profile of antibiotic detection (non exhaustive list)

Detection Limits (CCβ)

BEADYPLEX Analytical performance

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False positive rate

Bead n Blank samples n Screen positive False positive rate (%)

AMINOGLYCOSIDES_A 117 AMINOGLYCOSIDES_B 117 BETA-LACTAMS 107 LINCOSAMIDES 123 COLISTIN 122 MACROLIDES_A 113 MACROLIDES_B 113 SULFONAMIDES 107 PHENICOLS 118 TETRACYCLINES 117 (F)QUINOLONES 110 PLEUROMUTILINES 122 2 1,6

False positive rate < to those usually atributed to other screening methods for antibiotics

Parameter Condition 1 (control) Condition 2 Condition 3 SAMPLE

EXTRACTION

EXTRACTION VOLUME 1 ML 0,8 ML 1,2 ML AGITATION EXTRACTION TIME 10 MINUTES 5 MINUTES 30 MINUTES AGITATION SPEED 6,5 AGITELEC 4,5 AGITELEC 2,5 AGITELEC SAMPLE EXTRACTS STORAGE EXTRACTION + ANALYSIS 4°C ON

• 20°C ON

EXTRACTION METHOD AGITATION + CENTRIFUGATION STOMACHER

• ASSAY

PROCEDURE

NUMBER OF PLATE WASHING STEPS 3 1 2 TEST INCUBATION TEMPERATURE RT 4°C 37°C INCUBATION TIME COMPETITIVE STEP 30 MINUTES 15 MINUTES 60 MINUTES INCUBATION TIME REPORTERS STEP 15 MINUTES 5 MINUTES 30 MINUTES PLATE READING DELAY TEST + READING 1H DELAY ON DELAY

OTHER

REAGENTS (BEADS AND PRIMARY

BINDERS)

BATCH 1 BATCH 2 BATCH 3 OPERATOR A B C CYTOMETERS NOVOCYTE ACEA ACCURI C6 BD

• REAGENTS STABILITY

SAME DAY 3 MONTHS 4°C 6 MONTHS 4°C

BEADYPLEX Analytical performance

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Robustness

✓No impact of any of the evaluated parameters on assay performance ✓Extraction and analysis need to be performed the same day ✓Very robust method

BEADYPLEX Analytical performance

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Applicability

✓Porcine muscle ✓Bovine muscle ✓Poultry muscle ✓Fish (salmon and colin) ✓Milk Applicable to Honey Eggs FLEXIBILITY ✓Extraction procedure adaptation for different matrices ✓Cut-off modification for modulation of applicability

• r sensitivity

BEADYPLEX Analytical performance - Conclusions

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BEADYPLEX Analytical performance - Conclusions

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BEADYPLEX Developments

Hormones Sedatives Anti- inflammatory drugs Mycotoxines Food allergens Foodborne pathogenic bacteria Antibiotics residues

Allergens

FLOW CYTOMETRIC SANDWICH IMMUNOASSAY

BEADYPLEX Flow Cytometric Immunoassays – Assay principle

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BL RL

BEADYPLEX Flow Cytometric Immunoassays – Assay principle

Classification of particles Determination of assay signal intensity

B

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BEADYPLEX LOD/LOQ Range BLG 0,625 – 40 ppm Casein 0,625 – 40 ppm Egg 1,25 – 80 ppm Peanut 1,25 – 80 ppm Soy 1,25 – 80 ppm

Hormones Sedatives Anti-inflammatory drugs

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BEADYPLEX Hormones Sedatives Anti-inflammatory drugs

Corticosteroids Growth promoters

Screening multi-class and multi-residue of veterinary drugs in meat/milk or urine by FCIA Post-mortem test Ante-mortem test

Thank you for your attention

FCIA team

Alessandro Gallo Alexandre Monteforte Cédric Pety de Thozée Celia Suárez Pantaleón